The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Introduction
Fused-in Sarcoma (FUS) encodes a DNA/RNA-binding protein involved in numerous cellular processes including transcriptional regulation, RNA splicing, RNA transport and DNA repair. 1 Predominantly localized in the nucleus, FUS can shuttle between the nucleus and cytoplasm. 2 The FUS transcript is reported to have multiple domains including an N-terminal Gln-Gly-Ser-Tyr -rich region, an RNA-recognition motif, Arg-Gly-Gly repeat regions, a zinc finger motif and a highly conserved C-terminal NLS. [3][4][5] Variants in the FUS gene have been identified as potential causative factors for amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and frontotemporal lobar degeneration (FTLD). [6][7][8][9] FUS related mutations found in familial ALS/FTD patients are clustered in the C-terminal NLS, causing FUS to be mislocalized and accumulate as aggregates in the cytoplasm of neurons, initiating a pathway that contributes to neurodegeneration. 6,7 FUS function is reduced when aggregates form, but it is not yet known whether this initiates the pathogenic process or if the aggregates are pathogenic. 10 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.
Here, we compared the performance of a range of commercially-available antibodies for RNA-binding protein FUS and validated several antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of FUS properties and function.

REVISED Amendments from Version 1
To the introduction, we included frontotemporal local degeneration (FTLD) to the list of neurodegenerative diseases that the FUS gene may contribute to.
Any further responses from the reviewers can be found at the end of the article Results and discussion Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. [11][12][13][14][15] To identify a cell line that expresses adequate levels of FUS protein to provide sufficient signal to noise, we examined public proteomics databases, namely PaxDB 16 and DepMap. 17 HeLa was identified as a suitable cell line and thus HeLa was modified with CRISPR/Cas9 to knockout the corresponding FUS gene (Table 1).
For Western Blot experiments, we resolved proteins from WT and FUS KO cell extracts and probed them side-by-side with all antibodies in parallel 12-15 ( Figure 1).
For immunoprecipitation experiments, we used the antibodies to immunopurify FUS from HeLa cell extracts. The performance of each antibody was evaluated by detecting the FUS protein in extracts, in the immunodepleted extracts and in the immunoprecipitates 12-15 ( Figure 2). For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 18 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3).
In conclusion, we have screened FUS commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions. The underlying data can be found on Zenodo. 19,20 Methods

Antibodies
All FUS antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 21 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from

Antibody screening by Western Blot
Western Blots were performed as described in our standard operating procedure. 23  Antibody screening by immunoprecipitation Immunoprecipitation was performed as described in our standard operating procedure. 24 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μL of phosphate-buffered saline (PBS) (Wisent, cat. number 311-010-CL) with 0,01% triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μL of protein A-(for rabbit antibodies) or protein G-(for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.
HeLa WT were collected in HEPES buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 Â g for 15 min at 4°C. One mL aliquots at 1.0 mg/mL of lysate were incubated with an antibody-bead conjugate for~2 hours at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of HEPES lysis buffer and processed for SDS-PAGE and Western Blot on a 5-16% polyacrylamide gels.
Antibody screening by immunofluorescence Immunofluorescence was performed as described in our standard operating procedure. 12-15,18 HeLa WT and FUS KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 o C, 5% CO 2 . Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in PBS for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0,1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary FUS antibodies overnight at 4°C. Cells were then washed 3 Â 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 Â 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).
Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40Â oil objective (NA = 1.40). Analysis was done using the Zen navigation software (Zeiss

Kathleen Southern
Dear Ryota Hikiami, Thank you for your thorough review of this Data Note which analyses the performance of commercial antibodies for RNA binding protein FUS, a potential causative gene in many neurodegenerative diseases.
To answer your first point of feedback, literature reference 9, which refers to the publication by Van Langenhove et al., performs a mutational analysis of FUS in patients with cases of FTLD. To further elucidate this fact, we will be submitting a revised version with modified text to include FTLD in the list of neurodegenerative diseases potentially caused by FUS gene variants.
As for the second point, we always include a dataset in our reports, which includes all underlying raw data for the experiments performed (reference 20). This increases the transparency and reproducibility of our work. It also allows viewers to have a better understanding of the results and see what wasn't included in the figures. As for immunofluorescence, the dataset includes czi files of each antibody under the microscope.
We hope this provides the additional image coverage you were looking for.
Thank you again for your suggestions! Competing Interests: No competing interests were disclosed.